High-grade gliomas, also known as glioblastoma multiforme (GBM), are the most common and aggressive adult primary brain tumors. GBM patients have a minimal response to current therapies, including surgery, radiation and temozolomide chemotherapy. Most patients die within 14 months following diagnosis, emphasizing the urgent need for new therapies to combat this disease. GBM can be grouped into several molecular subtypes, including proneural, neural, mesenchymal and classical, based on distinct gene expression signatures. Of these subtypes, proneural GBM is particularly aggressive in younger patients, and most therapeutic approaches aimed at directly targeting tumor cells in this subtype have failed. In contrast to highly mutable tumor cells, non-cancerous stromal cells that support tumorigenesis in the tumor microenvironment (TME) represent genetically stable therapeutic targets. This means therapies targeted against the TME are less likely to result in the development of acquired resistance as a result of genetic changes in the stromal cells. Tumor-associated macrophages (TAMs) are an important cell type in the TME that correlate with increased tumor grade and poor patient prognosis in many cancers, including gliomas, suggesting important cancer-promoting functions. To examine the contribution of TAMs to glioma progression, we have used a genetically engineered mouse model of proneural GBM. We found that TAMs progressively increase with higher tumor grade in the mouse model, which parallels the increase observed during human glioma development. To investigate the functional significance of TAM accumulation, we designed preclinical trials in the proneural GBM model to therapeutically target colony stimulating factor-1 receptor (CSF-1R), which macrophages depend upon for survival and differentiation. CSF-1R inhibition as a monotherapy dramatically increased survival in these mice, and regressed established tumors after just 7 days. Macrophages were depleted in the normal brain, as we had expected, but not in gliomas of treated mice. Instead, glioma-secreted factors facilitated TAM survival in the presence of CSF-1R inhibitors. Interestingly, gene expression analysis of these surviving TAMs revealed a significant decrease in alternatively activated/ M2 polarization macrophage markers, and consistently, functional analyses revealed anti-tumorigenic phenotypes. Thus, TAM depletion is not strictly necessary for effective macrophage-targeted therapy. Rather, we propose that the presence of macrophage survival factors in the glioma TME not only enables TAMs to survive exposure to a CSF-1R inhibitor, but to be 're-educated' through this process, resulting in a striking anti-tumor response. Our preliminary data identifies TAMs as a promising therapeutic target for proneural gliomas, and establishes strong translational potential of CSF-1R inhibition in GBM. In this proposal, we will expand on these results to elucidate the mechanisms by which TAMs mediate glioma cell phenotypes, and determine how CSF-1R inhibitors interfere with this reciprocal communication to delay and block glioma progression. Our objectives are to elucidate how TAMs are initially educated by glioma cells, and then re-educated by CSF-1R inhibition in the glioma microenvironment. Next, we will investigate which glioma cell signaling pathways are enhanced by TAMs, and determine the downstream effectors that are critical to CSF-1R inhibition efficacy in vivo. Finally, we will determine whether gliomas develop resistance to CSF-1R inhibition, and identify the underlying mechanisms. To address these goals, we will employ multiple different methods including mouse glioma models and an extensive panel of co-culture assays to investigate communication between glioma cells, TAMs, and other cells in the glioma TME. We will combine analyses of known signaling pathways with the identification of novel targets using proteomics and expression profiling approaches. In addition to dissecting the underlying biological mechanisms by which TAMs promote glioma progression, the proposed experiments will also result in the development of therapeutic strategies for their specific inhibition, which if successful could ultimately be tested in the clinic. Collectively, these studies have important implications for future clinical consideration of CSF-1R inhibitors, and for other therapies that target the TME in cancer.